Novel 4-arylaminoquinoline-3-carbonitriles as Inhibitors of HIV-1 Reverse Transcriptase

Article abstract of DOI:10.1002/jhet.2914

This paper describes the synthesis of several new 4-arylaminoquinoline-3-carbonitriles derivatives. These were evaluated on the activity reverse transcriptase (RT) of HIV-1. Most of the synthesized compounds showed significant in vitro inhibition of RT en

Full text of DOI:10.1002/jhet.2914

Month 2017 Novel 4-arylaminoquinoline-3-carbonitriles as Inhibitors of HIV-1 Reverse
Transcriptase
Beatriz C. L. Melo,a Alice M. R. Bernardino,a Gustavo Polillo,a Helena S. Pereira,b Izabel C. P. Paixão,b
Michele S. Ribeiro,b and Julio C. Borgesa,c
*
aInstituto de Química, Programa de Pós-Graduação em Química, Departamento de Química Orgânica, Campus do
Valonguinho, Universidade Federal Fluminense, CEP 24020-141 Niterói, RJ, Brazil
bInstituto de Biologia, Programa de Pós-Graduação em Ciências e Biotecnologia, Departamento de Biologia Celular e
Molecular, Campus do Valonguinho, Universidade Federal Fluminense, CEP 24210-150 Niterói, RJ, Brazil
cInstituto Federal de Educação Ciência e Tecnologia do Rio de Janeiro, Campus Nilópolis, CEP 26530-060 Nilópolis, RJ,
Brazil
*
E-mail: julio.borges@ifrj.edu.br
Received January 2, 2017
DOI 10.1002/jhet.2914
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
This paper describes the synthesis of several new 4-arylaminoquinoline-3-carbonitriles derivatives. These
were evaluated on the activity reverse transcriptase (RT) of HIV-1. Most of the synthesized compounds
showed significant in vitro inhibition of RT enzyme, especially derivative 3h (IC50 = <8 μM). The
derivatives showed low-level cytotoxicity.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
phosphorylated to their triphosphate forms to act as
competitive inhibitors of the RT. The non-nucleoside
RT inhibitors (NNRTIs) are small hydrophobic
molecules that bind to an allosteric pocket of RT and
act as non-competitive inhibitor [8].
The human immunodeficiency virus type 1 (HIV-1) was
discovered in 1983 and is part of a subfamily of
retroviruses called Lentivirus. Human immunodeficiency
virus is the causative agent of acquired immunodeficiency
syndrome (AIDS), a life-threatening global fatal disease
[1,2]. An HIV particle, which is approximately 145 nm in
diameter and contains a linear single-stranded RNA
(ssRNA) genome encoding 15 mature viral proteins [3,4].
It destroys immune system leaving the victim vulnerable
to opportunistic infections, being responsible for millions
of deaths worldwide [5]. Many drugs have been
developed to try to inhibit the replication of HIV. Reverse
transcriptase (RT) is considered a key enzyme in the
replication cycle of retroviruses and has been widely used
as a tool to search compounds having antiviral potential
[6,7].
The two major classes of drugs that inhibit reverse
transcription are nucleoside analogues (NRTIs) and
nonnucleoside RT inhibitors (NNRTIs). The nucleoside
analogues (NRTIs) are compounds with a structure
like natural nucleosides and interact with the dNTP
binding site of the RT. All NRTI compounds are
known as 2,3-dideoxynucleoside analogues, with
similar mechanisms of drug action, and need to be
Quinoline moiety is present in many classes of
biologically active compounds such as antimalarial [9],
antibacterial [10], antifungal [11], antitumor [12], and
antiviral [13] agents. Our research group reported the
synthesis of various substances of pyrazolopyridine and
thienopyridine systems that showed antiviral activity
[14–16]. A series of 4-arylamino-1H-pyrazolo[3,4-b]
pyridine (I) inhibited approximately 40% of the RT
activity at 50-μM concentration [16]. Recent data show
the potential of quinoline derivatives to inhibit the
activity of enzyme RT of HIV-1 [17,18]. The quinolinic
ring was chosen based on the isosteric relationship
between it and the pyrazolo [3,4-b] pyridine ring (I),
which showed the excellent inhibition results on RT
shown above [16]. The arylamino group (also present in
compound I) was maintained, and structural
modifications were performed, such as variations among
the substituents present therein, so that a more efficient
screening could be performed. All 14 compounds
inhibited the RT-HIV-1 activity at micromolar
concentrations and showed no toxicity (Fig. 1).
© 2017 Wiley Periodicals, Inc.

B. C. L. Melo, A. M. R. Bernardino, G. Polillo, H. S. Pereira, I. C. P. Paixão,
M. S. Ribeiro, and J. C. Borges
Vol 000
can be readily made by methodology similar to the
Gould-Jacobs [20]. See Scheme 1.
Products were generally identified by 1H NMR, 13C
NMR, FT-IR spectroscopies, and mass spectrometry.
The 4-arylaminoquinoline-3-carbonitrile derivatives
were evaluated on their ability to inhibit HIV-1 RT
enzyme and the potential cytotoxic effect on human
T-cell lines transformed by HTLV-1 (MT2 cells). The
results are summarized in Table 1. Our results show
that derivatives 3a–n displayed low cytotoxicity
(CC50 ≥ 100 μM), with the exception of derivative 3j
that showed a higher cytotoxicity in lymphocyte (MT-2)
cells (CC50 = 0.09 μM). Derivatives 3a–n were tested
in RT inhibition assays and showed to be active as
inhibitors of HIV-1 RT (Table 1). The results
highlighted that derivative 3h (IC50 < 8 μM) turned out
to be more active. The derivatives 3a–g, 3i–k, and
3 m–n have also been showing promising IC50 values
ranging between 10 and 12 μM. In contrast, derivative
3l exhibited decreased activities towards the enzyme
(IC50 ≥ 50 μM). The results suggest that the 4-
Figure 1. Rational approach to the design of compounds 3a–n. [Color
figure can be viewed at wileyonlinelibrary.com]
RESULT AND DISCUSSION
The 4-arylaminoquinoline-3-carbonitrile derivatives
(3a–n) were prepared through the reaction of 4-
chloroquinolines-3-carbonitriles (2a, 2b) with anilines
using diethylene glycol as solvent (Scheme 1) [19–21].
The chlorine atom in the position 4 can be easily replaced
by nucleophiles, such as anilines in a nucleophilic
aromatic substitution. For other similar reactions, we
usually do not use solvent, and the reaction is completed
in approximately 4 h [19,20]. However, it was not
possible in the present case, due to higher melting points
of the intermediates 3a (199°C) and 3b (164°C). These do
not melt for the temperature required for such reaction
condition (120°C), and hence, the desired product could
not be obtained. Using the solvent diethylene glycol, the
reagents were easily solubilized, the optimum temperature
(120°C) for the reaction was reached, and the reaction
time was reduced to 1 h. Another advantage of this
solvent is easy removal from the reaction medium by
pouring into ice water. The 4-chloroquinolines (2a, 2b)
are readily prepared from 4-oxo-1,4-dihydroquinolines
(1a, 1b) [21]. The 4-oxo-1,4-dihydroquinolines, in turn,
arylaminoquinoline-3-carbonitrile derivatives are
a
promising target for studies in the search for new
substances with anti-HIV-1 activity, including in vitro
studies of their action mechanisms. Also, the relative
low cytotoxicity of the samples makes them potential
candidates for in vivo studies.
In summary, we report the synthesis of 4-
arylaminoquinoline-3-carbonitrile derivatives (3a–n).
Fourteen new compounds showed to be active as
Table 1
Cytotoxicity and HIV-1 RT inhibitory activities of compounds 3a–n.
Derivative
R
R0
CC50 SD(μM)a
IC50(μM)b,c
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
H
140 1.52
100 0.50
100 0.13
146 0.32
100 2.1
100 0.67
142 0.56
142 1.6
2.216 0.28
0.09 1.39
816 0.84
816 1.29
1.047 0.76
100 0.21
≤12
≤10
≤12
≤10
≤12
≤12
≤12
<8
≤12
≤12
≤12
≥50
≤12
≤10
30-F
30-Cl
30-Br
40-F
40-Cl
40-Br
H
Scheme 1. Synthesis of 4-arylaminoquinoline-3-carbonitriles 3a–n
(71–91%).
30-F
30-Cl
30-Br
40-F
F
F
40-Cl
40-Br
All values are the mean of three independent experiments.
aCellular toxicity of the 4-arylaminoquinoline-3-carbonitriles was
determined using T-cell line MT2. CC50 = Cytotoxic concentration that
reduced cell viability to 50%.
bThe inhibition of recombinant HIV-1 RT activity was performed with a
commercially available EnzChek Assay Kit (Molecular Probes) according
to the instructions of the manufacturer. IC50 = effective concentration that
inhibits 50% of HIV-1 RT.
cEfavirenz, an HIV-1 RT inhibitor used as positive control.
IC50 = 0.006 μM.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
4-arylaminoquinoline-3-carbonitriles as Inhibitors of HIV-1 Reverse
Transcriptase.
inhibitors of HIV-1 RT. The derivative that exhibited the
higher activity was 3h (R = F, R0 = H) with IC50 value of
8 μM. The compounds have low-level cytotoxicity and
represent a viable source of prototype antivirals. It was
identified chemical scaffold that may inform further
development of synthetic HIV-1 inhibitors and provide
new options for future anti-HIV therapies.
8-Chloro-4-(30-chlorophenylamino)quinoline-3-carbonitrile
(C16H9Cl2N3), 3c. Compound was obtained as a light yellow
solid. Yield 71%; mp 200°C; FT-IR (KBr) νmax/cmꢀ1: NH
3178, CN 2209, C═C/C═N 1580, 1559, 1522, 1475, 1402,
1286. 1H NMR (DMSO, δ in ppm): 8.87 (s, 1H, H-2), 8.53
(dd, J = 8.7 and 0.9 Hz, 1H, H-5), 7.75 (t, 8.7 Hz, 1H, H-6),
8.16 (dd, J = 8.7 and 0.9 Hz, 1H, H-7), 7.57–7.40 (m, 4H,
H-20, H-40, H-50, H-60), 10.28 (s, 1H, NH). 13C NMR (DMSO,
δ in ppm): 153.5, 90.1, 151.1, 121.3, 122.4, 126.7, 132.3,
144.8, 133.1, 141.1, 123.5, 133.4, 125.4, 130.6, 122.4, 116.4.
ESI-HRMS (M + H+): 315.0098.
EXPERIMENTAL
4-(30-Bromophenylamino)-8-chloroquinoline-3-carbonitrile
(C16H9BrClN3), 3d. Compound was obtained as a light yellow
solid. Yield 85%; mp 203°C; FT-IR (KBr) νmax/cmꢀ1: NH 3176,
Chemistry.
obtained using
The 1H NMR and 13C NMR spectra were
a Varian model Unity Plus spectrometer
1
CN 2209, C═C/C═N 1579, 1558, 1522, 1473, 1401, 1287. H
operating at 300.00 and 75 MHz, respectively. Infrared spectra
were recorded in Perkin-Elmer Spectrum One FT-IR
NMR (DMSO, δ in ppm): 8.88 (s, 1H, H-2), 8.53 (dd; J = 8.7
and 1.2 Hz, 1H, H-5), 7.76 (t, J = 8.7 Hz, 1H, H-6), 8.17
(dd, J = 8.7 and 1.2 Hz, 1H, H-7), 7.64 (t, J = 1.5 Hz, 1H,
H-20); 7.56–7.42 (m, 3H, H-40, H-50, H-60), 10.23 (s, 1H, NH).
13C NMR (DMSO, δ in ppm): 153.5, 90.0, 151.0, 121.7, 122.8,
126.7, 132.3, 144.8, 133.1, 141.1, 122.4, 121.2, 128.3, 130.8,
126.3, 116.4. ESI-HRMS (M + H+): 359.9489.
8-Chloro-4-(40-fluorophenylamino)quinoline-3-carbonitrile
(C16H9ClFN3), 3e. Compound was obtained as a light yellow
solid. Yield 70%; mp 215°C; FT-IR (KBr) νmax/cmꢀ1: NH
3352, CN 2204, C═C/C═N 1588, 1563, 1528, 1509, 1459,
1409, 1217. 1H NMR (DMSO, δ in ppm): 8.76 (s, 1H, H-1),
8.56 (dd, J = 8.4 and 1.2 Hz, 1H, H-5), 7.73 (t, J = 8.4 Hz, 1H,
H-6), 8.14 (dd, J = 8.4 and 1.2 Hz, 1H, H-7), 7.42–7.36
(m, 2H, H-20, H-60), 7.55–7.51 (m, 2H, H-30, H-50), 10.07
(s, 1H, NH). 13C NMR (DMSO, δ in ppm): 153.8, 87.7, 151.9,
120.4, 122.1, 126.4, 132.1, 144.6, 135.4, 133.1, 116.0, 128.1,
160.6, 128.1, 116.0, 116.4. ESI-HRMS (M + H+): 298.0349.
a
spectrophotometer in KBr disks. Melting points (m.p.) were
determined with a Fisher-Johns apparatus. All reagents and
solvents used were analytical grade. For fingerprinting ESI-MS
analysis, a hyBrid high-resolution and high-accuracy (5 ppm)
Micromass Q-TOF mass spectrometer (Micromass, Manchester,
UK) was used. The general conditions were as follows: source
temperature of 100°C, capillary voltage of 3.5 kV, and cone
voltage of 30 V. For measurements in the positive ion mode
ESI(+)-MS, 10.0 μL of concentrated formic acid was added
giving a final concentration of 0.1%. ESI-MS was performed by
direct infusion with a flow rate of 10 μL minꢀ1 using a syringe
pump (Harvard Apparatus). Mass spectra were acquired and
accumulated over 60 s, and spectra were scanned in the range
between 50 and 700 m/z. The equipment was calibrated with a
solution of phosphoric acid, permitting a resolution of less than
20 ppm.
Preparation of the 4-arylaminoquinoline-3-carbonitrile (3a–n):
1.0 mmol of 4-chloroquinolines (2), and 1.5 mmol of
corresponding aniline was stirred in 6.0 mL of diethyleneglycol
at 120°C for 1 h. Finaly, the mixture was poured in a beaker
with 100 mL of ice and water. The crystals formed were filtered
and recrystallized from ethanol.
8-Chloro-4-(40-chlorophenylamino)quinoline-3-carbonitrile
(C16H9Cl2N3), 3f. Compound was obtained as a light yellow
solid. Yield 74%; mp 235°C; FT-IR (KBr) νmax/cmꢀ1: NH
3340, CN 2204, C═C/C═N 1587, 1561, 1526, 1494, 1410,
1249. 1H NMR (DMSO, δ in ppm): 8.83 (s, 1H, H-2), 8.55
(dd, J = 8.5 and 1.2 Hz, 1H, H-5), 7.74 (t, J = 8.5 Hz, 1H,
H-6), 8.16 (dd, J = 8.5 and 1.2 Hz, 1H, H-7), 7.46 (dd, J = 8.7
and 2.1 Hz, 2H, H-20, H-60), 7.59 (dd, J = 8.7 and 2.1 Hz, 2H,
H-30, H-50), 10.10 (s, 1H, NH). 13C NMR (DMSO, δ in ppm):
153.6, 89.2, 151.3, 121.0, 122.3, 126.6, 132.3, 144.8, 133.1,
135.0, 126.2, 129.0, 138.4, 129.0, 126.2, 116.5. ESI-HRMS
(M + H+): 315.0098.
8-Chloro-4-phenylaminoquinoline-3-carbonitrile
(C16H10ClN3), 3a. Compound was obtained as a light yellow
solid. Yield 91%; mp 205°C; FT-IR (KBr) νmax/cmꢀ1: NH
3298, CN 2205, C═C/C═N 1583, 1559, 1531, 1494, 1482,
1408; 1H NMR (DMSO, δ in ppm): 8.78 (s, 1H, H-2), 8.58
(dd, J = 8.4 and 1.2 Hz, 1H, H-5), 7.76 (t, J = 8.4 Hz, 1H,
H-6), 8.15 (dd, J = 8.4 and 1.2 Hz, 1H, H-7), 7.57–7.37
(m, 5H, H-20, H-30, H-40, H-50, H-60), 10.08 (s, 1H, NH); 13C
NMR (DMSO, δ in ppm): 153.7, 88.7, 151.4, 120.9, 122.3,
126.2, 132.1, 144.7, 133.0, 139.1, 124.8, 129.1, 126.4, 129.1,
124.8, 116.4. ESI-HRMS (M + H+): 280.0510.
4-(40-Bromophenylamino)-8-chloroquinoline-3-carbonitrile
(C16H9BrClN3), 3g. Compound was obtained as a light yellow
solid. Yield 73%; mp 218°C; FT-IR (KBr) νmax/cmꢀ1: NH 3214,
1
CN 2206, C═C/C═N 1580, 1558, 1522, 1488, 1403. H NMR
(DMSO, δ in ppm): 8.85 (s, 1H, H-2), 8.55 (d, J = 8.5 Hz, 1H,
H-5), 7.72 (t, J = 8.5 Hz, 1H, H-6), 8.17 (d, J = 8.5 Hz, 1H,
H-7), 7.40 (d, J = 8.5 Hz, 2H, H-20, H-60), 7.77–7.69 (m, 2H,
H-30, H-50), 10.09 (s, 1H, NH). 13C NMR (DMSO, δ in ppm):
153.6, 89.5, 151.2, 117.9, 122.4, 126.6, 132.3, 144.8, 133.1,
138.8, 126.2, 131.9, 121.1, 131.9, 126.2, 166.4. ESI-HRMS
(M + H+): 359.9424.
8-Fluoro-4-phenylaminoquinoline-3-carbonitrile (C16H10FN3),
3h. Compound was obtained as a light yellow solid. Yield 78%;
mp 200°C, FT-IR (KBr) νmax/cmꢀ1: NH 3359, CN 2205,
C═C/C═N 1591, 1525, 1488, 1452, 1408, 1212. 1H NMR
(DMSO, δ in ppm): 8.72 (s, 1H, H-2), 8.42 (d, J = 8.1 Hz, 1H,
8-Chloro-4-(30-fluorophenylamino)quinoline-3-carbonitrile
(C16H9ClFN3), 3b. Compound was obtained as a light yellow
solid. Yield 76%; mp 212°C; FT-IR (KBr) νmax/cmꢀ1: NH 3301,
1
CN 2209, C═C/C═N 1585, 1560, 1528, 1490, 1448, 1402. H
NMR (DMSO, δ in ppm): 8.85 (s, 1H, H-2), 8.54 (dd, J = 8.7
and 1.2 Hz, 1H, H-5), 7.76 (t, J = 8.7 Hz, 1H, H-6), 8.14
(dd, J = 8.7 and 1.2 Hz, 1H, H-7), 7.32–7.16 (m, 4H, H-20,
H-40, H-50, H-60), 10.15 (s, 1H, NH). 13C NMR (DMSO, δ in
ppm): 153.5, 90.2, 151.1, 121.2, 122.4, 126.6, 132.3, 144.8,
133.1, 141.5, 110.9, 162.4, 112.2, 130.7, 119.9, 116.3.
ESI-HRMS (M + H+): 298.0372.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

B. C. L. Melo, A. M. R. Bernardino, G. Polillo, H. S. Pereira, I. C. P. Paixão,
M. S. Ribeiro, and J. C. Borges
Vol 000
H-5), 7.85–7.72 (m, 2H, H-6, H-7), 7.46–7.38 (m, 3H. H-20, H-4,
H-60), 7.58–7.52 (m, 2H, H-30, H-50), 10.09 (s1H, NH). 13C NMR
(DMSO, δ in ppm): 153.9, 89.0, 151.5, 121.6, 126.9, 119.3,
117.1, 158.0, 139.1, 139.6, 125.5, 129.6, 126.8, 129.6, 125.5,
117.0. ESI-HRMS (M + H+): 264.0902.
(DMSO, δ in ppm): 8.76 (s, 1H, H-2), 8.38 (d, J = 8.4 Hz, 1H,
H-5), 7.85–7.69 (m, 2H, H-6, H-7) 7.41 (d, J = 9.0 Hz, 2H,
H-20, H-60), 7.71 (d, J = 9.0 Hz, 2H, H-30, H-50), 10.11 (s, 1H,
NH). 13C NMR (DMSO, δ in ppm):153.2, 89.2, 150.7, 121.2,
126.5, 118.9, 116.7, 157.5, 138.7, 118.2, 126.4, 131.9, 138.6,
131.9, 126.4, 116.5. ESI-HRMS (M + H+): 343.9974.
8-Fluoro-4-(30-fluorophenylamino)quinoline-3-carbonitrile
(C16H9F2N3), 3i.
Compound was obtained as a light yellow
Biological and cytotoxity assays.
Escherichia coli strain
solid. Yield 73%; mp 220°C, FT-IR (KBr) νmax/cmꢀ1: NH
3358, CN 2208, C═C/C═N 1595, 1526, 1489, 1438, 1406,
1263, 1145. 1H NMR (DMSO, δ in ppm): 8.80 (s, 1H, H-2),
8.38 (d, J = 8.1 Hz, 1H, H-5), 7.87–7.73 (m, 2H, H-6, H-7),
7.31–7.26 (m, 2H, H-20, H-60), 7.60–7.53 (m, 1H, H-40),
7.23–7.17 (m, 1H, H-50), 10.11 (s, 1H, NH). 13C NMR (DMSO,
δ in ppm): 153.2, 89.9, 150.7, 120.2, 119.0, 116.7, 121.4, 111.1,
162.4, 112.4, 126.7, 120.1, 116.5. ESI-HRMS (M + H+):
282.0826.
BL21 (DE3) was used as a recipient for DNA transformations. E.
coli cells transformed with the plasmid containing RTp66 and
RTp51 HIV-1 gene were cultured in Luria-Bertani (LB)
containing ampicillin (100 μg/mL) under shaking, at 220 rpm at
37°C; overnight. This overnight culture was used as inoculum
for 1 L of LB medium containing 100 μg/mL of ampicillin.
Cells were grown for 6 h at 37°C with vigorous shaking, then
induced with isopropyl-b-D-thiogalactopyranoside (IPTG)
(1 mM) for 2 h. Cells were harvested by centrifugation (5000 g,
15 min), and bacterial lysates were prepared using a lysis buffer
(50 mM Tris–HCl (pH 7.9 at 4°C), 60 mM NaCl, 1 mM EDTA)
and using lysozyme/DNAse I treatment. Clarified lysates were
used for the isolation of the p51/p66 heterodimeric RT. The
active RT heterodimer was purified by using MagneHis™
Protein Purification System according to the manufacturer’s
instructions.
4-(30-Chlorophenylamino)-8-fluoroquinoline-3-carbonitrile
(C16H9ClFN3), 3j. Compound was obtained as a light yellow
solid. Yield 64%; mp 187°C, FT-IR (KBr) νmax/cmꢀ1: NH
3448, CN 2210, C═C/C═N 1594, 1582, 1528, 1498, 1432,
1
1206. H NMR (DMSO, δ in ppm): 8.81 (s, 1H, H-2); 8.38 (d,
J = 8.4 Hz, 1H, H-5), 7.87–7.75 (m, 2H, H-6, H-7), 7.58–7.39
(m, 4H, H-20, H-40, H-50, H-60), 10.11 (s, 1H, NH). 13C NMR
(DMSO, δ in ppm): 153.3, 89.8, 150.7, 126.7, 119.0, 116.8,
121.4, 125.7, 133.5, 123.8, 130.8, 122.7, 116.6. ESI-HRMS
(M + H+): 298.0494.
4-(30-Bromophenylamino)-8-fluoroquinoline-3-carbonitrile
(C16H9BrFN3), 3k. Compound was obtained as a light yellow
solid. Yield 79%; mp 200°C, FT-IR (KBr) νmax/cmꢀ1: NH 3211,
CN 2214, C═C/C═N 1593, 1579, 1526, 1500, 1475, 1433, 1406,
1209. 1H NMR (DMSO, δ in ppm): 8.81 (s, 1H, H-2), 8.37
(d, J = 8.1 Hz, 1H, H-5), 7.87–7.75 (m, 2H, H-6, H-7), 7.65
(t, J = 1.5 Hz, 1H, H-20), 7.57–7.50 (m, 3H, H-40, H-50, H-60),
10.10 (s, 1H, NH). 13C NMR (DMSO, δ in ppm): 153.2, 89.8,
150.7, 121.5, 122.7, 119.0, 116.7, 157.7, 126.8, 138.8, 125.7,
131.0, 123.8, 130.7, 126.5, 116.7. ESI-HRMS (M + H+): 343.0073.
8-Fluoro-4-(40-fluorophenylamino)quinoline-3-carbonitrile
HIV-1 RT inhibition. The effects of 4-arylaminoquinoline-
3-carbonitriles derivatives on the RT were evaluated using RT
EnzChek Assay Kit (Molecular Probes) according to the
manufacturer’s instructions. IC50 values were determined using
an Poly(A) ribonucleotide template annealed to a Oligo d(T)16
primer. To a 96-well solid black plate for fluorescence readings
was added 5 μL of increasing concentrations of derivatives,
followed by 1 μL of the enzyme (15–80 ng/mL) in reaction
buffer. The reaction was incubated at 25°C for 60 min, and
2 μL of 200 mM EDTA was added to each reaction. The
polymerizing activity was measured using a fluorometric assay
by adding 600 μL of PicoGreen in TE buffer (10 mM Tris–HCl,
pH 7.5, 1 mM EDTA) to the EDTA-terminated reaction
mixture. The reaction was incubated for 10 min on ice, in the
dark. The sample fluorescence was measured using a microplate
reader (Spectramax-M4 Molecular Devices) (ex. 480 nm, em.
520 nm). Efavirenz was used as a positive control. IC50 values
were determined using Prism5 (GraphPad Software). All assays
were performed in triplicate.
(C16H9F2N3), 3l.
Compound was obtained as a light yellow
solid. Yield 69%; mp 200°C, FT-IR (KBr) νmax/cmꢀ1: NH
3354, CN 2203, C═C/C═N 1589, 1567, 1528, 1488, 1468,
1418, 1407, 1213. 1H NMR (DMSO, δ in ppm): 8.69 (s, 1H,
H-2), 8.41 (d, J = 7.8 Hz, 1H, H-5), 7.83–7.69 (m, 2H, H-6,
H-7), 7.42–7.36 (m, 2H, H-20, H-60), 7.56–7.51 (m, 2H, H-30,
H-50), 10.04 (s, 1H, NH). 13C NMR (DMSO, δ in ppm): 153.4,
87.4, 151.5, 120.6, 118.7, 126.3, 116.5, 157.5, 120.7, 135.2;
128.1, 115.9, 160.7, 115.9, 128.1, 116.5. ESI-HRMS (M + H+):
282.0803.
Cell-based assay: cytotoxicity assays.
To evaluate the
cytotoxicity of 4-arylaminoquinoline-3-carbonitriles derivatives,
we used MTT assay accordingly to the manufacturer’s
protocol. Cytotoxicity was performed in the T-cell line MT2
(106 cells/well) treated with increasing concentrations of
derivatives at 37°C in a humidified 5% CO2 incubator for
72 h. After this, MTT solution (1 mg/mL) was added, and
cells were incubated for 4 h. After incubation, 100-μL stop
solution (0.04 N HCl) was added, and the absorbance was
determined in an automatic plate reader at 545 nm according
to the manufacturer’s instructions. CC50 values were
determined using Prism5 (GraphPad Software). All assays
were performed in triplicate.
4-(40-Chlorophenylamino)-8-fluoroquinoline-3-carbonitrile
(C16H9ClFN3), 3m. Compound was obtained as a light yellow
solid. Yield 75%; mp 248°C, FT-IR (KBr) νmax/cmꢀ1: NH 3352,
1
CN 2201, C═C/C═N 1587, 1524, 1483, 1410, 1213. H NMR
(DMSO, δ in ppm): 8.76 (s, 1H, H-2), 8.39 (d, J = 8.1 Hz, 1H,
H-5), 7.86–7.72 (m, 2H, H-6, H-7), 7.47 (d, J = 8.4 Hz, 2H,
H-20, H-60), 7.59 (d, J = 8.4 Hz, 2H, H-30, H-50), 10.08 (s, 1H,
NH). 13C NMR (DMSO, δ in ppm): 153.2, 88.9, 150.9, 121.1,
126.6, 118.9, 116.6, 157.5, 138.6, 138.2, 126.4, 129.0, 130.1,
129.0, 126.4, 116.6. ESI-HRMS (M + H+): 298.0529.
4-(40-Bromophenylamino)-8-fluoroquinoline-3-carbonitrile
(C16H9BrFN3), 3n. Compound was obtained as a light yellow
solid. Yield 75%; mp 245°C, FT-IR (KBr) νmax/cmꢀ1: NH 3353,
Acknowledgments. We thank Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), Coordenação
de Aperfeiçoamento de Pessoal Docente (CAPES), Fundação de
1
CN 2200, C═C/C═N 1588, 1524, 1482, 1407, 1212. H NMR
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
4-arylaminoquinoline-3-carbonitriles as Inhibitors of HIV-1 Reverse
Transcriptase.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet